1. Field of the Invention
The present invention is generally directed to biological compositions and methods for inhibiting phagocyte activation, such as polymorphonuclear neutrophil (PMN) activation, as exemplified by the inhibition of PMN chemotaxis, degranulation and superoxide production. The invention concerns the purification and characterization of novel peptide factors and the generation of inhibitory synthetic peptides and analogues with enhanced inhibitory activity. The peptides of the invention are contemplated for use as anti-inflammatory agents in the treatment of diseases such as rheumatoid arthritis, dermatitis, psoriasis or inflammatory bowel disease, and more particularly, in the treatment of lung diseases such as asthma and bronchitis.
2. Description of the Related Art
Pulmonary inflammation is associated with lung damage in numerous settings. Inflammation of lung parenchyma can cause severe oxygenation impairment (Swank & Moore, 1989) while bronchial inflammation often results in narrowing of the airway luminal diameter (Snapper & Brigham, 1984). Polymorphonuclear neutrophils (PMN), important cellular constituents of acute inflammatory processes, are attracted to areas of inflammation by chemotaxins released by resident cells (Sibille & Reynolds, 1990). However, because the lung is commonly exposed by inhalation to substances, such as bacteria and dust particles, that should trigger release of these chemotaxins, a mechanism to downregulate PMN influx is most likely present.
PMN activation occurs through an intricate cascade of chemical events that are initiated by binding of agonist to a plasma membrane receptor (Sklar, 1986). This is followed by coupling of a portion of the receptor to a GTP-binding protein with subsequent phosphorylation and activation of certain enzymes intermediate in the signal transduction process (Edelman et al., 1987). One enzyme that may be important in this process is phospholipase D (Cockcroft, 1989). The actions of phospholipase D cleavage products, I(4)P and I(4,5)P.sub.2 are not well characterized although certain studies have suggested they are important in the process of PMN activation. A recent study has suggested that a specific calmodulin-dependent protein kinase, myosin light chain kinase, may phosphorylate and activate phospholipase D in PMN (Kanaho et al., 1992).
PMN, although powerful antimicrobial cells, can also cause considerable tissue damage through release of toxic molecules. The influx of PMN into lung parenchyma may be associated with acute lung injury culminating in the adult respiratory distress syndrome. Alternatively, immigration of PMN into bronchi, occurring after a number of inhalational exposures, is associated with obstruction to airflow. The influx of PMN into pulmonary parenchyma or airways is known to occur through the production of numerous agonists that attract such cells (Sibille & Reynolds, 1990). However, little is known about the mechanisms which operate to attenuate PMN influx or to inhibit PMN activation in these settings.
Influenza A virus has been shown to be capable of deactivating PMN (Hartshorn & Tauber, 1988). Previous studies have documented that PMN oxidant production, degranulation, arachidonic acid release and chemotaxis are all attenuated by cellular infection with influenza A. However, the components of influenza A virus responsible for causing these effects have not been previously described.
In addition, various host cell-derived molecules have been described that inhibit neutrophil function in vitro. These molecules include elastase and cathepsin G, which inactivate chemotactic complement activation products, specifically C5a (Brozna et al., 1977), and alpha 1 antiproteinase which inhibits PMN chemotaxis to fMLP (Stockley et al., 1990). Neutrophils and monocytes have been noted to release a low MW factor, termed neutrophil immobilizing factor, that has an apparent Mr of 5,000 daltons, is trypsin digestible, and inhibits PMN chemotaxis to a variety of agonists (Goetzl & Austen, 1972). Lymphocytes release a protein in vitro that inhibits PMN chemotaxis (Klempner & Rocklin, 1983), and adenosine, released by platelets, is also known to inhibit certain parameters of PMN activation (Cronstein et al., 1990). Shephard et al. (1989) have demonstrated that high concentrations of nonpolar proteolytic peptide fragments of C reactive protein inhibit PMN chemotaxis and oxidant production. Finally, two cyclooxygenase products, prostaglandin E (Ham et al. 1983) and prostacyclin (Kainoh et al., 1990), inhibit PMN chemotaxis.
Molecules which inhibit phagocyte activation, such as inhibiting PMN function, have potential for use as anti-inflammatory and anti-proliferative agents. They would be particularly useful for treating a variety of lung diseases and disorders,-for example, asthma, bronchitis and acute lung injury. However, as phagocytes in general, and PMN in particular, are intimately involved in various immune and inflammatory responses, phagocyte inhibitors would also be suitable for reducing inflammation in other clinical settings, such as in the treatment of rheumatoid arthritis, inflammatory bowel disease, reperfusion cardiac damage after myocardial infarction, and various dermatological diseases such as psoriasis and dermatitis.
Unfortunately, the molecules documented to inhibit phagocyte and neutrophil function to date suffer from certain drawbacks that limit their potential for clinical use. For example, several of the potential PMN inhibitors described above have only been poorly characterized and have not been purified. In common with various cytokines, PMN inhibitors have a low abundance in biological systems and are therefore unlikely to be obtainable in quantities sufficient for clinical use from natural sources. Other effector molecules may not be suitable for clinical use due to, for example, high concentrations necessary to achieve inhibition, or a lack of defined biological specificity. Furthermore, although the mediators described above have anti-inflammatory properties in vitro, there are few investigations examining in vivo significance in the pulmonary setting.
Therefore, there currently exists in the art a need for the identification and characterization of phagocyte and PMN inhibitors, and particularly, those PMN inhibitors which can be produced in significant quantities. Elucidating the mechanism of action of a PMN inhibitor would provide further advantages, and the discovery of a molecule which inhibits PMN function at a central point in signal transduction would represent a significant breakthrough in the development of effective anti-inflammatory agents. The identification and production of a PMN inhibitor would lead to the development of anti-inflammatory agents particularly suitable for use in treating diseases associated with pulmonary inflammation, such as asthma, chronic bronchitis and acute lung injury.